Studies On The Electrochemical Biosensors Based On The Signal Amplification Technology

Use of chemo/bio sensor for environmental monitoring, food analysis and early diagnosis, has been a novel, attractive and hot topic in the current analytical chemistry. Electrochemical biosensors have received increasing attention due to their remarkable features such as high sensitivity, simple instrumentation, low production cost and promising response speed. In this thesis, based on the signal amplification technology, a series of novel electrochemical biosensors were developed for the detection of metal ions, DNA and protein.In chapter 2, based on the interaction between the T-Hg(Ⅱ)-T interaction and the amplification of bio-bar codes, a novel electrochemical biosensor for the sensitive detection of mercury ions has been designed. In the presence of Hg²⁺, 3'-end of the linker sequence hybridized with the electrode surface-tethered capture probe via the T-Hg(Ⅱ)-T interaction, and the gold nanoparticles functionalized reporter DNA subsequently hybridized with the linker, which amplified the signal. The results show that the fabricated sensor exhibits high sensitivity, good selectivity, and with a detection limit of 0.5 nM.In chapter 3, a novel electrochemical DNA sensor for detection of HIV-1 DNA based on the dual-amplification of Exonuclease III and the bio-bar codes was described. When target ssDNA hybridized with the electrode surface-tethered capture probe, with a 3'-overhang of nine nucleotides, Exonuclease III digested the capture probes, and released the intact target ssDNA. The released target ssDNA could hybridize with another capture probe, which created a repeated hydrolysis process, thus the amount of capture probe would be reduced. As a result, less bio-bar codes would hybridize, and the amount of [Ru(NH3)6]3+ adsorbed by the remained DNA on the electrode surface decreased, leading to the decrement of the current signal. The detectable dynamic range was from 100 pM to 100 nM.In chapter 4, a novel electrochemical immunosensor based on proximity dependent surface hybridization assay was described, which might creat a universal methodology for developing high-performance biosensors in sensitive detection of proteins. Taken PSA as a modle analyte, a pair of mono-antibodies recognized different antigenic determinants of PSA were used to covalently crosslink two oligonucleotides. One probe has a electroactive ferrocene-labeled tail sequence at the 3'-end that is complementary to the surface-tethered capture probe at 5'-end with a predesigned low melting temperature.Similary, the other probe has a tail sequence at the 3'-end that is complementary to the capture probe at 3'-end with a predesigned low melting temperature. Furthermore, the first probe at the backward tail sequence and the seconde probe at the foreward tail sequence have a short complementary region with a low melting temperature. Antibodies simultaneously bind to the antigene, two oligonucleotides tail sequences are brought into close proximity with their local concentration increased substantially to hybridize each other and allow the pair of tail sequences to hybridize together with the surface-tethered DNA strands. Then the ferrocene labels of the tail sequence are drawn close to the electrode surface and produce a detectable redox current. The PSA was determined in the range of 25 pg/mL to 2 ng/mL with a detection limit of 13 pg/mL and the immunosensor could be reusable.